Heme is essential for many cellular processes, including oxidative stress responses, detoxification, respiration, signal transduction, and can also serve as a regulatory molecule to affect gene expression. Both the control of heme synthesis and its regulatory function must be reconciled with the fact that heme and its precursors are toxic as free molecules, and iron may be a limiting nutrient. The broad objective of the proposed work is to elucidate the control of bacterial heme biosynthesis, and to reconcile regulatory functions for heme and its precursors with their toxicity in ceils. The heme biosynthetic pathway culminates with the insertion of iron into protoporphyrin catalyzed by ferrochelatase. The Irr protein from the bacterium Bradyrhizobium japonicum represses the pathway at an early step under iron limitation to prevent protoporphyrin synthesis from exceeding iron availability. Data suggest that Irr interacts directly with ferrochelatase, and responds to iron via the status of heme and protoporphyrin localized at the site of synthesis. In the presence of iron, ferrochelatase inactivates Irr, followed by heme-dependent Irr degradation to derepress the pathway. Under iron limitation, protoporphyrin relieves the inhibition of Irr by ferrochelatase, probably by promoting protein dissociation, allowing genetic repression. The proposal addresses the hypothesis that metabolic control of the heme pathway involves a direct input signal from a biosynthetic enzyme to a regulator to affect gene expression. Furthermore, heme can serve as a signaling molecule without accumulating freely in cells, irr gene homologs are found in numerous pathogens and symbionts of eukaryotes, suggesting lrr-type regulation in other bacteria. Three specific aims are proposed to address the model. 1. Elucidate the mechanism by which heme promotes iron dependent degradation of Irr. Irr is a conditionally stable protein that involves direct binding of heme to the protein. We propose experiments to address the role of heme with an emphasis on exploring heme-mediated oxidation and proteolysis. 2. Characterize interactions between ferrochelatase and Irr. This interaction allows Irr to respond to the status of heme synthesis and is essential for control of lrr degradation and activity. Experiments are designed to further characterize the interaction and to elucidate the molecular basis of the inhibition of lrr activity by ferrochelatase. 3. Characterize Irr regulatory function with respect to its DNA-binding properties, functional domains and gene targets.